Light Chain Dominant Quaternary Epitope Recognition and IgG3 Switching Drive Broad Dengue and Zika Virus Neutralization
Defining how antibodies achieve broad neutralization across antigenically related flaviviruses is essential for rational therapeutics and vaccine design. Here, we describe 3A06, a human monoclonal antibody that neutralizes dengue virus (DENV) serotypes 1-3 and zika virus (ZIKV). Notably, subclass switching to IgG3 exhibits expanded neutralizing activity against all four DENV serotypes. High-resolution cryo-electron microscopy (cryo-EM) analysis of 3A06 Fab bound to chimeric ZIKV virions shows that 3A06 engages a quaternary epitope spanning three E protomers. Functional analyses identify a major functional contribution from the light-chain, where somatic hypermutations are critical for neutralization potency, and reversion of improbable mutations substantially reduces neutralization and increases antibody dependent enhancement (ADE) at sub-neutralizing concentrations. Cryo-EM analyses of intact IgG3 in complex with chimeric DENV2 and DENV4 show that the conformational clexibility and extended IgG3 Fc-hinge enable bivalent engagement across E-protein rafts in multiple concigurations, overcoming geometrical constraints that limit binding in the IgG1 format. Together, these results define a distinct mode of epitope recognition relative to previously characterized E-dimer epitope (EDE)-specific broadly neutralizing antibodies (bnAbs), and demonstrate how IgG3 subclass architecture can expand virus neutralization breadth. These insights inform the rational engineering of antibodies and vaccines targeting quaternary epitopes on flaviviruses to overcome viral diversity.